1. Field of the Invention
The invention relates to a substantially purified form of migration inhibitory factor (MIF), a process of preparing the same and uses therefor.
2. Brief Description of the Background Art
Following activation, lymphocytes generally manufacture and secrete soluble substances variously referred to as factors, mediators or lymphokines, that affect the behavior of other cells. The factors can be divided into various groups according to the target cell they affect (macrophage, granulocyte, lymphocyte, or other). The three main effects of factors on macrophages are inhibition of macrophage migration, macrophage activation and macrophage chemotaxis. One of these factors, migration inhibitory factor (MIF), is the subject of the present invention.
MIF was the first mediator discovered. Lymphocytes from normal unimmunized animals do not produce MIF in measurable quantities, apparently because they are not stimulated in sufficient numbers by the antigen. For a similar reason, lymphocytes immune to one antigen will not produce measurable amounts of MIF when exposed in culture to another antigen. Normal lymphocytes, however, will produce MIF when activated by mitogens such as concanavalin A, for these substances activate a fair proportion of the lymphocyte population.
MIF has been demonstrated in most mammals, including humans. Its action is not species specific: for example, MIF of human origin inhibits macrophages obtained from guinea pigs.
In vitro MIF production can be demonstrated 4-6 hours after lymphocyte stimulation, and the stimulated cells continue to produce MIF for as long as four days, provided the stimulus persists. In vivo, the production of MIF--as well as that of most other mediators--is associated with delayed type hypersensitivity (DTH). Generally, the first exposure of a patient or animal to a given microorganism or to a non-bacterial protein produces no noticeable change, but the immune status of the recipient is clearly altered. The sign of this alteration is the fact that the recipient normally reacts differently to a second injection of the same antigen than it does to the first one. The first injection makes the animal hypersensitive to renewed antigen exposure; the major sign of this condition is the development of a characteristic skin lesion at the injection site--a lesion not seen after the first antigen exposure. Since the response to the second antigen inoculum is delayed by 24-48 hours, the reaction is referred to as a DTH. In humans, the sensitizing antigen derives from the microorganism responsible for the disease, for example tuberculin from Mycobacterium tuberculosis, typhoidin from Salmonella typhi, and abortus from Brucella, and sensitization occurs as a result of a chronic infection. DTH can manifest itself as either focal or systemic. The most characteristic focal manifestation of DTH is a skin reaction exemplified by the tuberculin test. Whole body (systemic) reactions occur particularly in cases in which large quantities of the antigen enter the blood stream. Symptoms of systemic reaction are fever, malaise, backache, pains in the joints, and reduction in the number of circulating lymphocytes. Severe cases of systemic reactions may result in shock, and even death, several hours after the antigen injection.
A rough scenario of the main events in DTH can be outlined as follows. The first exposure of an organism to the antigen results in sensitization of lymphocytes carrying the corresponding receptors on their surfaces. Following the second exposure to the antigen, the same diffuses through the skin and enters small veins. A few sensitized lymphocytes that have reached the site by chance recognize and bind the antigen, and this binding restimulates these cells. The restimulated lymphocytes release MIF. MIF acts on monocytes in the blood making them sticky and the sticky cells then adhere to the endothelial lining of the vein. MIF may also act on the endothelial lining, causing direct damage that may attract more monocytes. The invading monocytes force themselves through the endothelium of the vessel wall and enter the surrounding tissue. There, some of the cells transform morphologically into macrophages while others remain morphologically indistinguishable from blood monocytes. The result is an accumulation of monocytes and monocyte-like cells at the site of injection--the mononuclear cell infiltration. The macroscopic phenomena seen in DTH are swelling of the injection site caused by cellular infiltration, reddening caused by damage to the underlying blood vessels, and necrosis caused by enzymes and factors released by activated monocytes and lymphocytes.
One particularly relevant type of DTH is contact sensitivity, such as dermatitis brought about by inducing agents, including poiston ivy, poison oak, primrose, etc. MIF also plays an ubiquitous role in this type of DTH (see, generally, Klein, J., Immunology. The Science of Self-Nonself Discrimination, John Wiley & Sons, 1982, Chapters 7 and 12, at pp. 257-259 and 463-471).
Human MIF has been partially purified and characterized by gel filtration, immunoelectrofocusing (IEF) and sensitivity to proteases and neuraminidase (Weiser, W. Y., et al., Journal of Immunology, 126:1958 (1981)). The biochemical heterogeneity of MIF was found to relate to the duration of incubation of the lymphocytes and the antigen used. Klein, supra, indicates (see page 258, Table 7.2) that human MIF has a molecular weight of 25,000 daltons and is stable at 56.degree. C., migrates with human albumin on disc gel electrophoresis, is resistant to neuraminidase, and is sensitive to chymotrypsin. However, the material available prior to the present invention was not homogeneous as defined by strict sodium dodecylsulphate polyacrylamide gel electrophoresis standards, and no process of purifying MIF to such homogeneity was available.
Given the great importance of MIF in delayed type hypersensitivity, the need for a homogeneous standard usable in diagnostic immunology as well as possible therapeutic uses, the purification thereof was clearly a desirable goal.